JOURNAL OF VIROLOGY, Nov. 1993, p. 6339-63470022-538X/93/116339-09$02.00/0Copyright C 1993, American Society for Microbiology

In Vitro Expression of the Human Cytomegalovirus DNAPolymerase Gene: Effects of Sequence Alterations

on Enzyme ActivityLIN-BAI YE' AND ENG-SHANG HUANG' 2*

Lineberger Comprehensive Cancer Center' and Department of Medicine, Department of Microbiology andImmunology, and Curriculum of Genetics,2 University of North Carolina at Chapel Hill,

Chapel Hill, North Carolina 27599-7295

Received 7 June 1993/Accepted 27 July 1993

Genomic DNA of the Towne strain human cytomegalovirus polymerase (pol) gene (4.4-kb RsrII-NcoI segmentof the EcoRI J fragment) was cloned into plasmids containing either the T3 or the T7 promoter for in vitrotranscription-translation studies. The translation efficiency of unmodified pol cRNA was poor in this systemand could not be improved by capping. However, the efficiency could be enhanced by replacing the leadersequence with a 40-bp AT-rich sequence derived from an alfalfa mosaic virus, R4. pol cRNA directed thesynthesis of a 140-kDa polypeptide in a rabbit reticulocyte translation system. The in vitro-translated wild-typeenzyme possessed significant polymerization activity which could be stimulated by salt as could that of theauthentic enzyme purified from virus-infected cells. To study the critical domains of this enzyme, ninemutations were introduced into the pol gene around the conserved domains of eukaryotic polymerase byoligonucleotide-directed mutagenesis. Two constructs with mutations at amino acid residues 323 to 325(M32QS) and 725 to 726 (M7211) remained active, with partial loss of enzyme activity, while the enzyme

activities of other mutants with alterations at four domains located around amino acid residues 729 to 730(M73HN), 804 to 807 (M80 and DE80), 910 to 913 (M91 and DE91), and 962 to 964 (M96 and DE96) were

abolished. DNA template and triphosphate binding assays indicated that the mutation at 804 to 807 (conservedregion III) lost the ability to bind DNA template, and four mutants, M73HN (within conserved region II), M80(in region III), M91 (in region I), and M96 (around region V [962 to 964; amino acid sequence KKR]), failedto bind deoxyribonucleoside triphosphate. These data suggest that conserved region Ill is essential for DNAtemplate binding, while residues between conserved region II and V (725 to 964) are involved in triphosphatebinding.

Human cytomegalovirus (HCMV), a member of the herpes-virus family, is a ubiquitous human pathogen associated with a

wide variety of clinical manifestations. Like other herpes groupviruses, HCMV is able to enter latency and reactivate afterprimary infection. The outcome of HCMV infection often isasymptomatic, but severe and fatal infections are frequentlyencountered, in developing fetuses and immunocompromisedindividuals, such as transplant recipients and patients withAIDS (2, 13). HCMV has become a major opportunistic viralpathogen among organ transplant recipients and patients withAIDS. Thus, the development of anti-HCMV compounds hasbecome a priority in research on infectious diseases. In con-trast to herpes simplex viruses (HSVs), HCMV does notencode a virus-specific thymidine kinase (9). Although itencodes a protein phosphotransferase (UL97) which is able toweakly phosphorylate 9-(1,3-dihydroxy-2-propoxymethyl)gua-nine (DHPG, ganciclovir) in vitro, the substrate(s) for thisenzyme in virus-infected cells is unknown (27, 32). HCMV-specific DNA polymerase, therefore, is a prime target fordeveloping potent anti-HCMV compounds, and study of themechanism of action of this enzyme is warranted.

Purified HCMV DNA polymerase consists of two polypep-tides of 140 and 58 kDa (29). Comparison of this enzyme withthat of HSV-1 (14, 15, 32) and with some other a-like DNApolymerases (34) suggests that the 140 kDa peptide possessesthe core DNA polymerase activity. An accessory protein of

* Corresponding author.

HSV-1 DNA polymerase with a molecular size of 65 kDa(UL42) was able to increase the activity and processivity ofHSV-1 polymerase (10, 14, 33). Similarly, Ertl and Powell (7)demonstrated that the accessory protein of HCMV poly-merase, ICP36 (UL44), was able to physically and functionallyinteract with baculovirus-expressed 140-kDa core enzyme (8).Sequence analyses of the polymerase (pol) genes of the AD169and Towne strains ofHCMV have been done by Kouzarides etal. (23) and our laboratory (in preparation), respectively.Detailed comparison of the DNA sequences of the core

enzymes of both strains of HCMV with that of HSV-1 andEpstein-Barr virus reveals that more than 24% of their se-

quences are precisely conserved (23). Structural and functionalstudies of HSV-1 DNA polymerase as done previously by usingdrug-resistant mutants and in vitro site-directed mutagenesisresulted in information valuable for an understanding of thebiochemistry of the HSV-1 enzyme (5, 6, 11, 22, 30). On theother hand, although HCMV polymerase has been expressedin insect cells (7, 8), very limited information of a similarnature has been obtained for HCMV. We therefore made an

attempt, using in vitro site-directed mutagenesis and in vitrotranscription-translation approaches, to uncover the essentialdomains of HCMV DNA polymerase.DNA-dependent DNA polymerases, including those from

prokaryotes, eukaryotes, and viruses, contain several enzymat-ically active centers: (i) a DNA template binding site, (ii) a

primer binding domain, (iii) a 3'-5' exonuclease site, (iv) 5'-3'exonuclease activity, (v) a deoxynucleoside triphosphate(dNTP) binding site, and (vi) a DNA chain elongation domain

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HCMV EcoRI J Fragment ( 9.4 Kb )

TM C VO KUT E J N G R A S B LZX H I IJS F D W ST. 'I 111111 IIIi IIIIIIl '1I

pal Nol I 9.4 kbEI M13mp19EcoRi N,,.. - 2:= = = EcoRi

EcoRtoHindlEcoRi 4.5 Kb Hindillnp8Pol Digestion ~ l pE E- P1.Sb

EcoRI Rsrll HCol T3 4kb HindIllRsrII coRli

caRI Digestionsril Digestion Kienow ill-in, then

Kenow Fill InH' dil Dieto

Hi.dill NcoIEcoR

Hindlill

M13mp18EcoRI

FIG. 1. Schematic diagram of the location of the pol gene on the Towne strain HCMV genome and the construction of HCMVpol recombinantplasmids pEXpol, pApol-1, and pApol for in vitro expression of the HCMV pol gene. The HCMV pol gene was located within a 9.4-kb EcoRI Jfragment of HCMV DNA. Transcription direction is indicated by arrows. Standard procedures were used to construct HCMV DNA polymerasegene clones as described in Materials and Methods. The rationale for cloning the HCMVpol gene first in both M13mpl8 and M13mpl9 is to obtaintwo orientations of pol gene constructs (left). The scheme on the right shows the procedures for inserting the full-length pol reading framerecovered from mpl8pol into pGEM EX-1 at a position downstream of the T3 promoter (pEXpol) and into pGEM1 (from pA9) at a positiondownstream of the AMV R4 leader sequence (pApol and pApol-1). In pEXpol, the HCMVpol leader sequence between EcoRI (AluI) and RsrIIwas retained as in pApol-1; in pApol, this HCMV leader sequence was removed by RsrII digestion. Plasmid pA9 (18) was used to generate apGEM-1 vector with AMV R4 leader sequence to construct pApol-1 and pApol. In this case, pApol-1 and pApol both contain the AMV R4 leadersequence downstream of the T7 promoter. RB, retinoblastoma cDNA.

(21). Comparison of the amino acid sequences of these ox-likeDNA polymerases shows that they share regions of strikingsequence similarity. Furthermore, these conserved regionsoccur in the same sequential order within the enzyme mole-cules (34), suggesting that they share common basic molecularstructures and that the active domains within the conservedregions are functionally related among polymerases of variousorigins. Therefore, previous studies of prokaryotic as well aseukaryotic enzymes, particularly that of HSV-1, provide valu-able information and guidelines for designing experiments tostudy the biochemistry and active domains of HCMV-specificDNA polymerase.The purpose of this publication is to report the expression of

the HCMV pol gene in vitro, the effects of HCMV pol leadersequence on RNA translation, and the identification of severalessential and active moieties of the core enzyme of HCMVDNA polymerase via the application of site-directed mutagen-esis and in-frame deletion mutation. In vitro transcription-translation studies were used to analyze the effects of thesemutations on HCMV DNA polymerase activity.

MATERIALS AND METHODS

Construction of HCMV pol gene recombinants. HCMVTowne strain (passages 36 to 40) was used in this study. Thelocation of the EcoRI J fragment containing the HCMV DNApolymerase gene is shown in upper left corner of Fig. 1. M13recombinant phages and plasmids carrying the HCMV polgene were constructed by standard methods and are shown onthe left side of Fig. 1. In brief, Towne strain HCMV DNA waspurified from extracellular virus as described previously (17).An HCMV DNA genomic library was constructed in pBR322by using EcoRI-restricted HCMV DNA fragments (2). A9.4-kb HCMV EcoRI J fragment which contained the pol genewas recovered from plasmid pHD10 by EcoRI digestion.Because the 5' end of the HCMV pol gene lacks an appropri-ate enzyme site, the intact pol DNA fragment was constructedin two subcloning steps. The first involved 5'-half manipula-tion. The EcoRI J fragment was digested with BglII, and the2-kb BglII fragment containing the glycoprotein B gene and 5'half of the pol gene was recovered and inserted into the

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HCMV DNA POLYMERASE 6341

M13mpl9 BamHI site to create recombinant phage M13mpl9Bgl II. The M13 mpl9Bgl II replicative form (RF) DNAwas digested with PstI and BamHI. A 1.2-kb PstI-BamHI DNAfragment was recovered and partially digested with AlIul togenerate a 0.74-kbAluI-BamHI DNA fragment, spanning frompositions -23 relative to the initiation codon to +719 of thepol gene. This 0.74-kb AluI-BamHI fragment was then insertedinto M13mpl9 between the SmaI and BamHI sites. Afteridentification of both flanking ends by DNA sequencing, theresultant recombinant phage was named M13 mpl9pol-1. TheM13 mpl9pol-1 RF DNA was cut with Ncol (at position +650)and ligated to the 3.8-kb NcoI subfragment (spanning +651 to+4422 of the pol gene), generated from NcoI digestion of thegenomic EcoRI J fragment. The orientation of insertion was

confirmed by PstI digestion and DNA sequencing. Thus, thefull HCMVpol reading frame starting from 23 bp upstream ofthe initiation codon to the second poly(A) signal at the 3' endwas cloned into M13mpl9; this final recombinant was desig-nated mpl9pol. The mpl9pol RF DNA was digested withEcoRI and HindlIl, and the resulting 4.5-kb DNA fragment ofthe pol gene was then cloned into mpl8 between the EcoRIand HindIll sites to generate mpl8pol. In mpl9pol, the polDNA sequence is in the antisense direction relative to theunique HindIll site, while in mpl8pol, it is in the sense

direction (Fig. 1, bottom left).To express the pol gene in vitro, a recombinant plasmid,

pEXpol, carrying the pol gene under the control of the T3RNA polymerase promoter was constructed (Fig. 1, upper

right corner). Plasmid pGEM EX-1 DNA (Pharmacia) was

digested by EcoRI and Hindlll, and the 4.5-kb EcoRI-HindIIIDNA fragment of the pol gene from mp I8pol was inserted intopGEM EX-1 between the EcoRI and HindIll sites to yieldpEXpol. Thus, in pEXpol, the pol gene is located immediatelydownstream from the T3 promoter.

The efficiency of the in vitro translation of pol cRNA madefrom pEXpol DNA was very poor (see Results). We specu-

lated that the GC-rich 5' untranslated leader sequence of theauthentic HCMV pol gene retained in the pEXpol RNAtranscript might limit the translation efficiency in a rabbitreticulocyte system or that factors other than those existing inthe reticulocyte lysate may be needed to overcome this hand-icap. Therefore, we constructed another series of recombinantplasmids for in vitro translation. pA9, a pGEM1 derivative witha retinoblastoma cDNA insert (a kind gift from W. H. Lee),contains 40 bp of high-AT alfalfa mosaic virus (AMV) R4leader sequence downstream from the T7 promoter (18). Atthe 3' end of the R4 leader sequence, there is an AhaII (Narl)site. We subsequently used this site as the base to engineer theHCMV pol recombinants containing R4 leader sequence.

Plasmid pA9 was partially digested with AhaII (Fig. 1, right),filled in with Klenow enzyme to create blunt ends at bothterminals, and then digested with HindIll. The desired 2.8-kbAhaII-HindIII DNA fragment with the T7 promoter and AMVR4 leader sequence was recovered and used as a vector toconstruct two kinds of plasmids with a difference in length ofthe remaining leader sequences downstream from R4 (Fig. 1,lower right corner). In the first construction, the mpl8pol RFDNA was digested with RsrII and filled in with Klenowfragment to create a blunt end before being cut with HindlIl.The resulting 4.5-kb DNA HindlII-RsrII fragment of the pol

gene was ligated to the linearized 2.8-kb AhaII-HindIII vectorto yield the recombinant plasmid pApol. In pApol, the pol gene

was downstream from a T7 promoter, and the original 5'noncoding leader sequence of HCMV pol was removed toposition -7 relative to the translation initiation site by RsrIIdigestion and replaced with a stretch of high-AT sequences

Leader Sequence

T3 Alul RsrilpgEX polMcS 16 7(AT )

T7 AMV Leader Alul Rsrllmm~ I pApol-IMCS 16 7(ATG)

T7 AMV Leader Rsril________L pApol

7(ATG)

Enzyme ActivitypM 32P dAMP IncorporatedU q 1.0 1.5

[0.01

012.5

0.1813.8

1.0I2 0.5 1.0

Relative Quantity ot Peptide Synthesized

FIG. 2. Comparison of the in vitro translation efficiencies ofpEXpol, pApol-1, and pApol. Two micrograms each of in vitro-synthesized cRNAs from linearized pEXpol, pApol-1, and pApol wassubjected to in vitro translation as described in the text. Translationproducts were subjected to SDS-PAGE and DNA polymerase activityassays. The plasmid arrangements of constructs are shown on the left.Five microliters each of translation lysate was used for DNA polymer-ization assays and SDS-PAGE analysis. The amount of Pol peptidesynthesized was measured by using the intensity of the 140-kDapeptide obtained in each reaction and expressed as relative activity,using the intensity of the pApol 140-kDa peptide as 1.

from the AMV R4 leader sequence (5'-GTTl7l`TTATTTTTAATTTTCl[T[CAAATACTTCCATCGGCGGTCCGCTATG-3 ).We prepared another construction, pApol-l (Fig. 1, middle

right), in which the leader sequence between All and RsrII(5'-CTGTCAGCCTCTCACG-3', -23 to -8 from the initia-tion codon) were preserved downstream from the AMV R4sequence. The differences between pApol-l and pApol is thatpApol-1 has an additional 16 bases(Al/lI to RsrII) downstreamfrom the AMV R4 sequence (Fig. 2).

Oligonucleotide-directed mutagenesis. Oligonucleotideswere synthesized by using an Applied Biosystems DNA syn-thesizer and purified by electrophoresis in 20% acrylamide-8M urea gels. Full-length oligonucleotides were recovered fromgels and phosphorylated by polynucleotide kinase reaction.The sequences of oligonucleotides used for mutagenesis are asfollows: M32QS, 5'-GCA CAC GCACGC GAT CAG AATGAC AAT-3'; M7211, 5'-GTG GGC CAT GAG GAG GGAAGG GTA-3'; M73HN, 5'-GTA GCA GAG GTC GTC GGCCAT GAT-3'; M80, 5'-TTT GAG CGC CAT CAG TAC AATGCC GAG CAA CAT ACG-3'; DE80, 5'-TTT GAGCGCCAT GAGCAA CAT ACG-3'; M91, 5'-GCG GAC AAACAC GCG GAC CGC GCC CCC GTA GAT GAC-3'; DE91,5'-GCG GAC AAACAC CCC GTA GAT GAC-3'; M96,5'-ThTT GCC GAT GTA AAG TATCGA GCA GAT CATCAT-3'; and DE96, 5'-TTT GCC GAT GTA GCA GAT CATCAT-3'.

Oligonucleotide-directed mutagenesis was performed as de-scribed by Kunkel (26). A pol DNA KpnI-HindIII fragment(+2168 relative to the HindIll site after the second polyade-nylation signal) was cloned into the M13mpl8 KpnI-HindIIIsite to create mpl8pol K-H, in which single-stranded phageDNA was used as a template for the mutagenesis reaction(except for M32QS). Uracil-containing single-stranded recom-binant phage DNA was obtained by growing the M13mpl8recombinant phage in Escherichia coliCJ236 in the presence of50 [tg of uridine per ml in 2x YT medium (10 g of yeastextract, 16 g of tryptone, and 5 g of NaCl per liter) and wasused as a template for the mutagenesis reaction. To facilitatemutation, two 5'-phosphorylated oligonucleotides, a mu-

tagenized oligonucleotide of pol sequence and an oligonucle-otide of M13 sequence, were used together to anneal with the

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template. The mutagenesis and synthesis reaction was carriedout with Klenow fragment at 16°C overnight in the presence ofthe four dNTPs (200,uM each) and T4 DNA ligase (1 U per

reaction). The end product of this reaction was extracted withphenol-chloroform and used to transform E. coli JM107 com-

petent cells. The recombinant mutants were screened, andDNA sequences were confirmed by DNA sequence analysis bythe dideoxy-chain termination method. The pol DNA frag-ments with the correct mutations were isolated and used toconstruct the pGEM series of plasmids for in vitro transcrip-tion.

In vitro transcription and translation. Wild-type and mu-

tant plasmids, both pEXpol and pApol cloned in pGEMvectors, were linearized with HindlIl. Linearized DNAs were

purified by low-melting-point agarose gel electrophoresis.DNAs were recovered from the gel, extracted with phenol-chloroform, and then precipitated with ethanol. In vitro tran-scription was carried out by using T7 RNA polymerase for thepApol DNA template and with T3 RNA polymerase forpEXpol constructs. For uncapped transcription, the reactionmixture contained 10 [LI of 5 x transcription buffer (40 mMTris-HCl [pH 7.9], 6 mM MgCl2, 2 mM spermidine), 2 jig oflinearized DNA, 2.5,lI (10 mM each) of ATP, GTP, CTP, andUTP, 50 U of RNasin, 5 RI of 100 mM dithiothreitol, 5 jil of10-mg/ml bovine serum albumin, and 50 U of RNA poly-merase; the final volume was made up to 50 pI with distilledH20. The reaction mixture was incubated at 37°C for 1 h. Thetemplate DNA was digested with DNase I at 37°C for 15 min.For capped transcription, 5 pI of 10mMm7GpppG was addedto the reaction mixture and the final concentration of GTP was

reduced to 50 ,uM. The synthesized RNA was purified byphenol extraction and ethanol precipitation in the presence of1 M ammonium acetate and then dissolved in diethyl pyrocar-

bonate-treated H20. The purified RNA was sized and quanti-tated on 1.2% agarose-formaldehyde gels. Four microliters ofRNA ladders with sizes ranging from 0.24 to 0.95 kb (1 jig/pA)was applied in parallel for size measurement by gel electro-phoresis.The RNA was translated in 50 [lI of reaction mixture

composed of 10 jiCi of [35S]methionine, 2 jg of RNA, and 35plI of reticulocyte lysate (Promega Biotec) at 30°C for 60 min as

instructed by the supplier. Aliquots were then taken for DNApolymerase activity assay and for protein analysis by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prestained protein molecular weight markers (Bio-Rad, Richmond, Calif.) were used for size measurement.

DNA polymerase assay. DNA polymerase activity assays

were carried out as described previously (29). In brief, thereaction mixture, in a 50-pA volume, contained 50 mM Tris-HCl (pH 8.0), 70 mM ammonium sulfate, 10 mM MgCl2, 5 jig

of activated calf thymus DNA, 10 jiM each dGTP, dYTP, anddCTP, 2.0 ,uCi of [cx-32P]dATP (3,000 Ci/mmol; New EnglandNuclear), and 5 jil of translation product. Polymerase reactionmixtures were incubated at 37°C for 30 min. The reaction was

stopped by the addition of 100 jil of 20% ice-cold trichloro-acetic acid containing 0.4% acid hydrolyzed yeast RNA and0.02 M sodium pyrophosphate, set in ice for 15 min, and thenfiltered onto a Whatman GF glass filter. Filters were washed

five times with 5 ml of 5% trichloroacetic acid. Acid-precipi-

table radioactivity was measured by liquid scintillation count-

ing. Three samples from each transcription-translation reac-

tion were assayed for enzyme activities.DNA template and deoxyribonucleoside triphosphate bind-

ing assays. For the DNA template binding assay, purified

pRc/RSV plasmid DNA (5.2 kb; Invitrogen, San Diego, Calif.)was linearized by HindIll digestion and nicked with pancreatic

DNaseI (10-4 U/100,ug/ml; at room temperature for 15 min,followed by heat inactivation at 70°C for 15 min). The nickedpRc/RSV DNA was used as the template for detecting theability of the enzyme to bind DNA template. Ten microliters of[35S]methionine-labeled DNA polymerase and 10,ug of nickedDNA template were mixed in polymerase assay buffer and keptin ice for 30 min. The reaction mixture was then loaded onto a5-ml 10 to 50% (wt/vol) sucrose gradient and centrifuged for3.5 h at 46,000 rpm in a Beckman SW50.1 rotor. Fractionswere collected from the bottom, and the radioactivity in eachfraction was measured.

For the deoxyribonucleoside triphosphate binding assay, thecold in vitro-translated polymerase, wild type or mutant, wasmixed with 1 RCi of [a-32P]dATP in 40 RI of polymerasereaction buffer and incubated at 37°C for 30 min. The reactionmixture was then loaded onto a 5-ml 10 to 30% (wt/vol)sucrose gradient. After centrifugation in a Beckman SW50.1rotor for 15 h at 46,000 rpm at 4°C, fractions were collectedfrom the bottom of the tube, and the radioactivity in eachfraction was measured.

RESULTS

Construction of HCMV pol templates and in vitro expres-sion. The 4.5-kb HCMV DNA fragment containing the com-plete HCMV pol gene coding region and two polyadenylationsignal sequences at the 3' end was cloned into M13mpl8,M13mpl9, and pGEM EX-1. The resultant constructs,mpl8pol, mpl9pol, and pEXpol, respectively, each retainedthe original 23-bp GC-rich noncoding leader sequence up-stream of the initiation codon. In pEXpol, the pol gene wasdownstream from the T3 promoter. After linearization byHindIll, the pEXpol DNA as well as all nine mutated pEXpolDNAs were transcribed in vitro with T3 RNA polymeraseunder the conditions for capping as well as uncapping. They allyielded almost the same quantity of 4.5-kb RNA products. Nodistinguishable difference in size among the transcriptionproducts of wild-type pEXpol and mutated pol genes wasfound when the products were sized and quantitated onagarose-formaldehyde gels (data not shown). The size of the4.5-kb RNA product corresponds with the predicted size of thewild-type pol DNA transcript. The purified RNAs were sub-jected to in vitro translation using rabbit reticulocyte lysate inthe presence of [35S]methionine. The translation mixtures wereassayed directly for enzymatic activity and were subjected toprotein analysis by SDS-PAGE. Very little polymerase activitywas found in the translation mixtures, including RNAs fromwild-type and nine mutated pol gene constructs, both cappedand uncapped. Also, no detectable polypeptide with a molec-ular size of 140 kDa, like that of native HCMV DNA poly-merase core enzyme, was observed when the reaction productswere subjected to SDS-PAGE. On the other hand, the controlbrome mosaic virus RNA, provided with the kit, translatedvery well under similar conditions.The linearized pApol-1, pApol, and nine mutated pol gene

plasmids (pApol mutants), all with AMV R4 leader sequence,were then transcribed in vitro with T7 RNA polymerase. Thein vitro transcription efficiency for all constructs indicatedabove did not show significant variation. On the other hand, asshown in Fig. 2, a significant difference in translation wasobserved between pApol and pApol-1.

In vitro-transcribed RNAs from pApol and pApol mutantswere then translated in rabbit reticulocyte lysate as describedabove. The major translation product from the wild type andmutants has a molecular size of 140 kDa in SDS-PAGE, a sizecorresponding with that of the core enzyme purified from

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HCMV DNA POLYMERASE 6343

A BX d - I w 0. 41f% M uM 0W-

|.......IF

205-

140 _- _

116

205-140*

116-

80- -

...."..E ..

80

49-

FIG. 3. SDS-PAGE analysis of in vitro translation products ofwild-type HCMV pol and its mutants. Two micrograms of wild-type(WT) or mutant pol cRNA was translated in rabbit reticulocyte lysatein the presence of [35S]methionine; 5 RI of each translation mixturewas analyzed by SDS-PAGE (10% polyacrylamide gel). The arrow

indicates the 140-kDa Pol peptide. Molecular weight markers are

shown in kilodaltons on the left. Panels A and B represent two

separate experiments.

HCMV-infected cells (29) and with the predicted size of theHCMVpol open reading frame (Fig. 3). The relative yields of140-kDa translation product from the wild type and themutants are almost identical. To determine the enzyme activityof the 140-kDa polypeptide synthesized in the programmedtranslation mixtures, each reaction mixture was directly as-

sayed for enzymatic activity in the presence of 0.07 M ammo-

nium sulfate. Relatively high DNA polymerase activity fromwild-type pApol was detected in the reticulocyte reactionmixtures. This enzyme activity could be stimulated approxi-mately onefold by the addition of 35 to 70 mM ammoniumsulfate compared with the level without ammonium sulfate.This characteristic is similar to that of the authentic HCMVDNA polymerase extracted from virus-infected HEL cellsreported previously (16, 29).

Site-directed mutation of the HCMV pol gene and its effectson enzymatic activity. Oligonucleotide-directed mutagenesisand the in vitro transcription-translation system provide a

direct approach to study the functional domains of HCMVpolymerase. Nine pol mutations, either point or deletionmutations, were introduced into the HCMV pol gene aroundregions homologous to five conserved regions of eukaryoticpolymerase. The nucleotide sequences and amino acid alter-ations for each mutation are summarized in Fig. 4A. Three ofthem (DE80, DE91, and DE96) are in-frame deletion mutantswith deletions of three to four amino acid residues. DE91contains a deletion of four amino acid residues, 910 to 913(DTDS), in the most conserved regions among the a-like DNApolymerase family (region I; Fig. 4B). In M91, these acidicDTDS residues (residues 910 to 913, deleted in DE91) were

changed into the more hydrophobic and basic GAVR. DE80contains a deletion of residues 804 to 807 (DKEQ) withinconserved region III. In M80, charged moiety DKEQ was

changed to the more hydrophobic and noncharged GIVL.DE96 contains a deletion of charged residues 962 to 964(KKR) around conserved region V. In M96, the chargedsequence KKR was changed to hydrophobic noncharged SIL.The other three mutants (M32QS, M7211, and M73HN)contain site mutations which result in alterations of two aminoacid residues each. In M32QS, residues Q and S at positions323 and 325 are changed to hydrophobic L and A, respectively.

In M7211, two I residues at 725 and 726 are changed to two Lresidues. Mutant M73HN contains two altered residues at 729and 730, in which the H and N residues are changed to acidicDD. Both M7211 and M73HN contain mutations within onelong stretch of conserved region II of the DNA polymerase ofthe herpesvirus family.

All nine mutated pol genes were expressed in vitro alongwith the wild type, and programmed translation mixtures wereassayed to determine the effects of these mutations on enzymeactivity (Fig. 3 and 4B). The levels of synthesis of the 140-kDapolypeptide, which represent HCMV pol, in all translationreactions were nearly equal, as estimated by radioautograms ofintensity (Fig. 3). This finding implies that these mutations didnot affect pol gene peptide synthesis in in vitro transcriptionand translation in pGEM and reticulocyte systems, respec-tively. Two mutants, M32QS and M7211, retained one-half toone-third, respectively, of the wild-type enzyme activity (Fig.4B). Modification of both isoleucine residues at 725 and 726(M7211, in region II) to leucines resulted in only a 40%decrease in enzyme activity, while almost all of the enzymeactivity was abolished when H and N residues at 729 and 730(M73HN) were changed to acidic DD. The other six constructs(DE80, DE91, DE96, M80, M91, and M96) lost nearly allenzyme activity. These data suggest that conserved regions I,II, III, and V are essential domains for HCMV polymeraseactivity. Mutagenesis study of conserved region VI was notincluded in this first-phase investigation because it has a lowlevel of homology with that of HSV-I polymerase (6 of 17amino acid residues).DNA template and triphosphate binding ability. We used

sucrose gradient sedimentation analysis to study the abilities ofthe in vitro-translated wild-type and mutated HCMV poly-merases to bind DNA template and deoxyribonucleosidetriphosphate. Figure 5A shows the results of DNA templatebinding assays of wild-type and mutated enzymes. In this study,[35S]methionine-labeled enzyme was mixed with nicked andlinearized plasmid DNA molecules. A specific radioactive peakcontaining plasmid DNA and polymerase complexes was foundaround fraction 6 (from fractions 5 to 8) of a sucrose gradientof the wild-type enzyme. This radioactive peak could beabolished by the addition of an excessive amount of nonlabeledwild-type enzyme (not shown). Similar peaks appeared insucrose gradients of other mutants except that of M80. Thisresult indicates that among the mutations studied, only thedeletion or mutation at amino acid residues 804 to 807 (withthe change from DKEQ to GIVL) results in the loss oftemplate binding ability. Controls, including (i) reticulocytelysate with DNA template and (ii) wild-type enzyme withoutthe addition of DNA template, did not produce a sedimenta-tion peak like that of the wild-type polymerase.

Figure SB shows the results of the triphosphate bindingassay. In this study, the cold in vitro-translated wild-type ormutant DNA polymerase was mixed with [x-32P]dATP andsubsequently subjected to sucrose gradient centrifugation. Thewild type and two mutants (M32QS and M7211) were able tobind [32P]dATP and exhibited a radioactive peak aroundfraction 7 of each gradient, while the remaining four mutants(M73HN, M80, M91, and M96) did not show this ability. Thisresult suggests that domains around residues 729 to 730 (HN),804 to 807 (DKEQ), 910 to 913 (DTDS), and 962 to 964(KKR) of HCMV DNA polymerase are critical for triphos-phate binding. The reticulocyte translation control did not givethis specific radioactive peak in the sucrose gradient.

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952 9g9WT: 5'- GAT GAC ATT GTC ATT CAG ATC TCG TGC GTG TGC TAC GAG ACG GGA GGA- 3'

318 Q323 s I'5 _3

M32QS: CTG GCG2158 j A 2202

WT: 5'-AGC CTC TAC CCT TCC ATC ATC ATG GCC CAC AAC CTC TGC TAC TCC -3720 17s 1 r 734

M7211: CTC CTCL 725 L 726

2170 2217WT: 5-TCC ATC ATC ATG GCC CAC AAC CTC TGC TAC TCC ACCTG TTG GTG CCG -3'

724 Hr29 N 739

M73HN: GAC GACD729 D 750

2395 2442

WT: 5- CGT CGT ATG TTG CTC GAC AAG GAA CAG ATG GCG CTC AAA GTA ACG TGC - 3'

799 8o14DW4 K E Q 8C7DE 80: delete >

GGC ATT GTA CTGM 80: G804 V LOW

2713 2760WT: 5'- CGG GTC ATC TAC GGG GAC ACG GAC AGC GTG TTT GTC CGC TTT CGT GGT - 3'

905 D 910T D S 913 920

DE 91: delete >GGC GCG GTC CGC

M 91: G910 A V R913

BAA

2899 2916

WT: 5'- CTT ATG ATG ATC TGC AAG AAA CGT TAC ATC GGC AAA GTG GAG GGC GCC -3'957 962 964 g2

K K Rdelete

DE 96: >TCG ATA CTT

M 96: S I L

200 400 600 800 1000 1210M32QS

M7211

M73HN *

M80 m i

DE80EO

M91 .

DE91 ==r

M96

DE96 ==l

WT g L

NH2 [-fl CGOOHIV 11 VIl III V

EXPRESSIONProtein Activity+ 4.39

8.07

+ 0.71

: 0.58

+ 0.81

_0.40

++++

0.71

0.45

0.39

13.70

FIG. 4. Summary of nucleotide sequences and amino acid alterations in various HCMV pol gene mutants (A) and mutation and deletionanalyses of the HCMV DNA polymerase gene (B). A schematic diagram of amino acid (AA) residues 1 to 1242 is shown at the top of panel B.Sites of mutations (in M32QS, M721I, M73HN, M80, M91, and M96) and deletions (in DE80, DE91, and DE96) are indicated by solid bars andbroken lines, respectively. The six conserved regions defined by Wong et al. (34) and their locations relative to the HCMV Pol peptide arepresented at the bottom of panel B. DNA polymerization activities of in vitro expressed peptides are summarized at the right, expressed aspicomoles of [32P]dAMP incorporated into acid-insoluble material in 30 min at 37°C.

DISCUSSION

Many eukaryotic genes have noncoding regions of variouslengths located upstream of the translation initiation codon(24). These 5' untranslated leader sequences play very impor-tant roles in the rate limiting as well as the efficiency oftranslation both in vivo and in vitro (25). The molecular basisof the role of 5' untranslated sequences in regulating mRNAtranslation is not well understood. Jobling and Gehrke (19)found that the composition of the untranslated sequence had a

marked effect on the quantity of protein synthesized. Thissequence can be replaced by a heterologous leader sequence toincrease expression without changing the antigenic or biolog-ical properties of the encoded protein (18). In this study, the

translation level of RNA from pApol deleted of the high-GCleader sequence was about six to eight times higher than thatof the RNA from pApol-1 with the authentic high-GC leadersequence, as estimated by enzyme activity in translation mix-tures and by the autoradiographic intensity of the 140-kDa Polpeptide from each translation. This result suggests that anAT-rich composition in the untranslated leader sequences isessential for maximal activity in HCMV pol RNA translation.It also implies that the GC-rich region in the leader sequencewhich was deleted in pApol inhibits pol RNA translation. Thetranslation efficiency may relate to the secondary structure orhigh GC content of the untranslated leader rather than theunique nucleotide sequence in this case. The formation of

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HCMV DNA POLYMERASE 6345

A6-

4

2NO 40

40

X 20 4m

2I.

323

2

3

2

2 4 6 8 10 12bt top

Fraction Number

B

2 4 6 8 10 12bt top

Fraction Number

FIG. 5. DNA template and deoxyribonucleoside triphosphate binding analysis of HCMV polymerase mutants. (A) DNA template bindingassay. Ten micrograms of linearized and nicked pRc/RSV DNA and 10 p.l of [35S]methionine-labeled DNA polymerase were mixed in polymeraseassay buffer, kept in ice for 30 min, and subjected to 10 to 50% (wt/vol) sucrose gradient centrifugation at 46,000 rpm for 3.5 h at 4°C in a BeckmanSW50.1 rotor. Fractions were collected from the bottom of the tube for the measurement of [35S]methionine radioactivity. Controls includereticulocyte lysate with DNA template (Cl) and wild-type (WT) enzyme without the addition of DNA template (C2). (B) dATP binding assay. Tenmicroliters of in vitro-translated unlabeled polymerase was mixed with 1 p.Ci of [a-32P]dATP in 40 ,ul of polymerase reaction buffer, incubated at

37°C for 30 min, and subjected to 10 to 30% sucrose gradient centrifugation in a Beckman SW50.1 rotor for 15 h at 46,000 rpm at 4°C. Fractionswere collected from the bottom of the tube for measurement of 32P radioactivity. The translated product from reticulocyte lysate without additionof pOI cRNA was used as the control (C).

hairpin structures by GC-rich sequences within the leader or

between the leader and other regions may inhibit the initiationof translation (25). Other unique factor binding domainswithin leader sequences, such as sequences for cap-bindingprotein or ribosome recognition and binding, as well as a

domain for the binding of immediate-early gene products,could contribute to the regulation of translation (31).

In HCMV-infected HEL cells, pol RNA can be detected as

early as 5 h postinfection, but viral DNA replication does notreach the peak level until 72 h postinfection. This can beattributed to the low-level expression of the HCMV DNApolymerase gene early in the infection. The effect of the 5'leader of pol RNA on translation may be one of the factorsresponsible for the low-level expression of DNA polymeraseactivity at earlier times. A similar result was found in the studyof translational regulation of HSV pol. Yager et al. (35)demonstrated that expression of the HSV DNA polymerasegene in vivo was regulated at the stage of translation. The HSVpol mRNAs were relatively abundant in the cytoplasm ofinfected cells at 6 h postinfection. However, only a smallfraction of mRNAs were associated with large polysomes.Comparison of the synthesis of this Pol protein with that ofanother HSV delayed-early polypeptide, thymidine kinase,shows that the molar ratio of thymidine kinase and Polpolypeptide synthesis per transcript is greater than 18:1 (35). A144-base GC-rich leader sequence with the hairpin structureencompassing the HSV pol initiation codon has been impli-cated as causing this low efficiency of HSVpol translation at anearly stage of infection.HCMV DNA polymerase shares conservative functional

domains with a group of eukaryotic DNA polymerases. On thebasis of amino acid sequence homology, Wong et al. (34)identified six highly conserved regions among phage, viral, andeukaryotic DNA polymerases. In HCMV DNA polymerase,these conserved regions are located within amino acid residues379 to 1100. The major DNA polymerization domains of theHCMV enzyme could be within the region between residues710 and 1100. This region contains highly charged amino acidresidues, such as Glu, Asp, Asn, Arg, and Lys, and sulfur-containing Cys and heterocyclic His that are needed to interactwith DNA, substrate, Mg2+, and other proteins or subunits ofDNA polymerase. In fact, all mutations that tend to loseenzyme activity fall within this region.Mutant M73HN contains two substituted residues (from HN

to DD) in conserved region II. It shows a lack of DNApolymerization activity as measured in our assay conditions.Results from DNA template and triphosphate binding studiesindicated that mutant M73HN retained the ability to bindDNA template, but its triphosphate binding ability was lost.This region is located within the largest stretch of conservedregion II of the herpesvirus family, and the N residue is one ofthe most conserved amino acid residues within this region. Incontrast to M73HN, mutant M7211, containing two substitutedresidues (from II to LL), was only partially affected by theII-to-LL alteration. This can be explained by the minor alter-ation in the hydrophobic region; amino acid residues of thesame nature might not significantly affect the enzyme activity.On the other hand, the mutation identified as a single residuechange at 719 and 724 in region II of HSV-1 pol led to theresistance of HSV replication to drugs such as phosphonoace-

N

v-

ErI,

Q

J00

m

00

0

E

a

z

0

0

Cla10

10

9

7

5

9

7

549

7

57

5659

7

55

4

C

WT

M32QS

M72 II

M73HN

M80

M91

M96

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6346 YE AND HUANG

tic acid (11, 22). This result implies that this domain may bedirectly or indirectly involved in the recognition of substratebinding and that the specific amino acid residue for thisfunction is critical. With respect to the functional domain inregion III, mutation of this region with an in-frame deletion or

alteration in residues DKEQ to GIVL (804 to 807), such as

DE80 and M80, destroyed almost all enzyme activity in an invitro assay. DNA template binding studies showed that mutantM80 lost its ability to bind nicked DNA template (Fig. 5A), asdid mutant D80 (data not shown), while the remaining mutantsstill possessed template binding ability. In the triphosphatebinding assay, we also failed to detect the ability of M80enzyme to bind triphosphate. One of the characteristics ofconserved region III which distinguishes it from other con-served regions is that most of the highly conserved residues are

basic or amide-containing amino acids, such as Q, K, and N. Itis possible that region III is involved in DNA template bindingvia the interaction of charged moieties with DNA template. Inthe case of the HSV enzyme, a cluster of mutations inconserved region III (at residues 813, 815, 821, and 842)developed resistance to deoxyribonucleoside triphosphate an-

alogs (11, 12). This region also contains Q-, N-, R-, K-, andC-rich segments. It is therefore postulated that this region isinvolved in the interactions with both DNA and triphosphatesubstrates. Aspartate and glutamate residues are directly in-volved in the binding of metallic ions. Metal ions (Mg2+,Mn2+, and Zn2+) have been show to be critical for substratebinding by a direct interaction with the phosphodiester bond(1, 4). The amino acid DKEQ domain deleted in mutant DE80may also be involved in the hydrolysis of the phosphodiesterbond of the dNTP (1, 4).DE91 and M91 have deleted and mutated DTDS domains,

respectively, within the most conserved region I of eukaryotica-like DNA polymerase. M91 was able to bind DNA templatebut unable to bind the substrate dATP. Delurue et al. (3)suggested that region I located in a loop at the end of a

hairpin and an Asp residue interact with Mg2+ to form part ofan NTP binding site. Site-specific mutations in HSV-1 Pol andhuman adenovirus type 2 DNA polymerase in the highlyconserved region I have been studied extensively by Dorskyand Crumpacker (6) and Joung et al. (20), respectively. Theirresults indicated that each of residues GDTD within thisregion was essential for enzyme function. Mutants G885R,D886N, T887K, and D888A of HSV-1 pol lacked polymeriza-tion activity and failed to be stimulated by the 65-kDa DNA-binding protein. However, another mutant (S889A) within thisregion had decreased but detectable enzyme activity, and itsactivity was able to be stimulated by the presence of the65-kDa DNA-binding protein (6). In case of adenovirus type 2polymerase, the change from S1015A in region I was alsoassociated with a decreased but still detectable level of enzymeactivity when measured by a pol DNA-binding protein assay

(20). Recently, Marcy et al. (30) demonstrated that mutationsin HSV Pol region I, F891C, F891Y, and V892M, exhibiteddrug resistance to phosphonoacetic acid and acyclovir andhypersensitivity to aphidicolin. These results implied that theGDTDS domain within region I contributed directly to theformation of a substrate dNTP recognition and binding site.Another in-frame deletion mutant (DE96) or point mutant

(M96), at positions 962, 963, and 964 within conserved regionV (residues 971 to 988), containing three deleted or changedamino acid residues (KKR to SIL) also lost almost all enzy-

matic activity. The results from DNA template and triphos-phate binding assays indicated that these mutants possessedDNA binding activity but failed to bind dATP.Among these six groups of mutation constructs, four,

M73HN, M80, M91, and M96, were unable to bind to dNTPsubstrate. This finding suggests that amino acid domainscontributing to triphosphate binding are not confined to oneregion but are distributed throughout the central and C-termi-nal portions of the peptide. This observation is further sup-ported by the fact that a series of HSV mutants that wereresistant or hypersensitive to nucleoside analogs have beenmapped throughout regions I, II, III, V, and VI. More recently,HCMV mutants resistant to ganciclovir, HPMPA {(S)-1[(3-hydroxy-2-phosphonylmethyl)propyl]adenine}, and HPMPC{(S)-1 [(3-hydroxy-2-phosphonylmethyl)propyl]cytosine} andhypersensitive to thymidine arabinoside were mapped at con-served region IV on the N-terminal side of the molecule (28).It is possible that amino acid domains of these mutations arealso involved in recognition and binding of NTP or in deter-mination of the tertiary structure of the binding site.

It is virtually impossible to delete or change a residuewithout any change in protein structure and hydrophilicity.However, two-dimensional structure analysis by computerreveals that these mutants have not changed drastically thestructure and hydrophilic nature of the Pol peptide beyond themutated regions. We are not able to perform detailed three-dimensional structure analysis of protein by computer methodsat the present time. Therefore, to further define the functionsof these conserved regions, it is essential for us to express andpurify various mutated Pol proteins for functional assay undera variety of conditions, including the presence of other proteinsassociated with the DNA replication complex.

ACKNOWLEDGMENTS

We thank Wen-Hwa Lee for plasmid pA9 and Shu-Mei Huong andJinrong Gao for technical assistance.

This investigation was supported by Public Health Service grantsCA21773, CA15036, and A112717 from the National Institutes ofHealth.

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